PROJECT SUMMARY During normal mammalian development, the hematopoietic system undergoes controlled maturation. Driving this process, hematopoietic stem and progenitor cells (HSPCs) execute dramatic transitions in their defining characteristics. These include changes in self-renewal, lineage biases, repertoires of differentiated cells produced, and possibly even potency. This process is of critical significance in the biology of age-specific blood disorders, hematopoietic stem cell transplantation, normal aging, and efforts to engineer of patient- specific hematopoietic cells for use in therapy. Our work and that of others has implicated the highly conserved heterochronic Lin28b/let-7 microRNA regulatory axis in specification of the developmental state of HSPCs. We have shown that repression of the biogenesis of let-7 microRNAs by Lin28b defines erythroid- biased fetal myeloerythroid progenitor output, while downregulation of Lin28b expression during development to adulthood permits the robust myelopoiesis characteristic of the mature bone marrow. The Preliminary Data in this proposal supports the emerging paradigm that individual CMPs are imbued with a single lineage destiny at their genesis (i.e., unipotency) that is dictated by their transcriptional state. This proposal tests the hypothesis that during maturation of the hematopoietic system, populations of HSPCs evolve in their heterogeneous single cell composition in order to effect developmentally necessary changes in self-renewal, potency, and lineage restricting branch points; and that the Lin28b/let-7 axis plays a central role in regulating this process. The Specific Aims of this proposal are to: 1) investigate the role of Lin28b/let-7 in regulating the changes in lineage potency that occur over developmental time in vivo; 2) directly connect the activity of the Lin28b/let-7 axis to lineage outcomes; and 3) evaluate the mechanisms by which Lin28b/let-7 modulates the gene regulatory networks that specify developmental state in HSPCs. I am currently a clinical fellow in Pediatric Hematology-Oncology at Boston Children's Hospital. I have proposed a five-year career development plan that will build upon the foundation of my clinical background in Pediatric Hematology as well as my prior research experience in developmental hematopoiesis to establish a career as an independent investigator/physician-scientist at a major academic center. Under the combined mentorship of Dr. George Q. Daley, M.D., Ph.D. and Dr. Benjamin L. Ebert, M.D., Ph.D., established leaders in research in normal and diseased hematology, I propose rigorous investigation into the fundamental mechanisms underlying normal developmental maturation of hematopoietic progenitor cells. The K08 award will provide me with the necessary protected time to execute the proposed studies and career development plan within the five-year time frame.